Synthetic polypeptides and receptor molecules derived therefrom and methods of use

ABSTRACT

Antigens, immunogens, inocula, antibodies, receptors, diagnostic methods and systems relating to tuberculosis mycobacteria are disclosed. Each of the compounds, compositions, methods or systems contains about 40 residues, or an antibody containing site that immunoreacts with such a polypeptide. The polypeptide includes the thirteen or fourteen amino acid reside sequence (AlaLysValAsnIleLysProLeuGluAspLysIleCys) or (CysAlaLysValAsnIleLysproLeuGluAspLysIleCys). When linked to a carrier and introduced in an effective amount into a mammalian host, the polypeptide is capable of inducing production of antibodies that immunoreact with an antigen to a tuberculous mycobacterium.

The U.S. Government has rights in this invention pursuant to a grantawarded by the National Institutes of Health.

This is a division of application Ser. No. 765,048, filed Aug. 12, 1985,now U.S. Pat. No. 4,689,397.

TECHNICAL FIELD

The present invention relates to immunogens, antigens, inocula,antibodies, methods and systems useful in the detection, diagnosis andtreatment of diseases involving mycobacterial infections.

BACKGROUND OF THE INVENTION

Mycobacteria have long been recognized as bacterial pathogens of man andcontinue to produce devastating illness, particularly in developingcountries. World Health Organization, Bull. WHO, 61, 779 (1983).Tuberculosis is caused by respiratory infection with Mycobacteriumtuberculosis (M. tuberculosis) and currently afflicts about 30 millionpeople worldwide with an annual mortality of about 3 million.

Crude bacterial antigen preparations have been used for immunodiagnosticand immunoprophylactic purposes. The tuberculin test developed by Kochin 1881 was the first immunodiagnostic test used in man. Tuberculin, anM. tuberculosis filtrate of complex but poorly defined composition, iscurrently used as a delayed-type cutaneous hypersensitivity (DCH) orskin test antigen to detect prior exposure to the pathogen. Seibert etal., Am. Rev. Tuberc., 69, 585 (1954). Unfortunately, the utility oftuberculin is limited both by its lack of specificity and by itsinability to distinguish between active disease, prior sensitization bycontact with M. tuberculosis or cross-sensitization to othermycobacteria.

Bacillus Calmette-Guerin (BCG), an avirulent strain of Mycobacteriumbovis (M. bovis), is currently used as a live vaccine to protect againsttuberculosis in man. Calmette, A., J. Am. Med. Assoc., 96, 58 (1931).While BCG has been effective in reducing the incidence of tuberculosisin Western Europe [Medical Research Council, Bull. WHO, 46, 371 (1972)],it has recently been found to be ineffective in a major trial in India[World Health Organization, WHO Tech. Rep. Ser., 651 (1980)].

A basic problem with the crude antigen preparations that are now used todetect mycobacterial infections is that the antigen preparations oftenreact positively with several different mycobacterial species. This, ofcourse, complicates diagnosis and the selection of an appropriatetreatment regimen. A reagent that specifically evokes an immune responseagainst a particular mycobacterial species would be beneficial in thediagnosis and management of mycobacterial infections.

The advantages of the use of a defined polypeptide antigen in a DCHreaction are numerous. For example, in the case of mycobacterialinfections, since the amino acid residue sequence of the polypeptidecorresponds to a portion of a protein that is specifically expressed inthe tuberculous mycobacterial species, the polypeptide may be a usefulreagent for specifically detecting a tuberculous mycobacterial infectionand thereby circumventing the cross-reactivity problems associated withthe currently-used skin test antigens. Moreover, the polypeptide may bechemically synthesized to eliminate the need to grow large cultures of apathogenic organism for the production of skin test antigens.

The polypeptide may also be used in the detection or prevention(vaccination) of mycobacterial infections. In fact, an inoculumcontaining the polypeptide could replace tuberculin or PPD (purifiedprotein derivative) as the antigen of choice in DCH or skin tests forthe detection of tuberculosis in humans.

While the general concept of preparing synthetic antigens (immunogens)and using them to induce antibodies of predetermined specificity hasbeen described, there remains a large area of this technology thatcontinues to defy predictability. There are at least two reasons forthis. First, a synthetic antigen (immunogen) does not necessarily induceantibodies that immunoreact with the intact protein in its nativeenvironment. Second, the natural antibodies of a host to a naturallyoccurring immunogen, such as a viral protein, rarely immunoreact with apolypeptide that corresponds to a short linear portion of theimmunogen's amino acid residue sequence. This latter phenomenon isbelieved to be the result of short linear polypeptides lacking requiredsecondary and tertiary conformational structures.

Much of the work on the binding of peptide by antibody made to proteinsis summarized in a review by Benjamini, E., et al., Current Topics inMicrobiology and Immunology, 58, 85 (1972). The role of peptidestructure in antibody binding has been emphasized by Goodman, J. W.,Immunochem 6, 139 (1969).

Most of the studies that involve the effects of changes in the sequenceof peptides on antibody binding have been interpreted as indicating thatthe structure of the antibody combining site plays a predominant role.The effect of sequence and structural changes in these studies isintermixed and difficult to segregate. Some of these studies can equallywell be explained by structural changes in antigen effecting thebinding.

Antibody response at the molecular level involves binding of an antigenof defined sequence (primary structure) and in a defined conformation(secondary and tertiary structure). Immune response to protein antigenshas traditionally been interpreted as being directed against primary,secondary or tertiary structure of the protein.

This classification scheme may have some validity for proteins that havea well defined overall structure at physiological temperatures andsolutions. However, its validity is in doubt for peptide antigens thathave a more dynamic structure.

SUMMARY OF INVENTION

The present invention contemplates synthetic polypeptides capable ofinducing the production of antibodies that immunoreact with an antigento a tuberculous mycobacterium. The polypeptides contain about 13 toabout 40 amino acid residues and includes the amino acid residuesequence, written from left to right and in the direction ofamino-terminus to carboxy-terminus, represented by the formula:

AlaLysValAsnIleLysProLeuGluAspLysIle. The polypeptides can includecysteine (Cys) residues at one or both of the amino-terminus andcarboxy-terminus.

The polypeptides are capable, when linked to a carrier and introduced inan effective amount into a mammalian host, of inducing production ofantibodies that immunoreact with an antigen to a tuberculousmycobacterium. The invention also includes the pharmaceuticallyacceptable salts and antigenically related variants of the polypeptides.

The polypeptides may also be capable of immunoreacting with humanantibodies induced by a natural antigen to a tuberculous mycobacterium.

The present invention also contemplates synthetic multimers containing aplurality of joined synthetic polypeptide repeating units wherein atleast one of the repeating units is a polypeptide as described above.The polypeptide repeating units may be joined in a head-to-tail mannerby amide bonds. Alternatively, the synthetic polypeptide monomers may bejoined by other than amide bonds to form a polymeric multimer such asthrough the use of intramolecular, interpolypeptide cysteine disulfidebonds.

In another embodiment, an effective amount of a polypeptide of thisinvention is used in a physiologically tolerable diluent to form aninoculum capable of inducing antibodies that immunoreact with an antigento a tuberculous mycobacterium. In addition to being used for theproduction of antibodies, an inoculum of this invention may be used as avaccine in humans as a means for inducing active immunity tomycobacterial infections.

In still another embodiment, a receptor molecule is contemplated thatcontains an antibody combining site that is capable of immunoreactingwith an antigen to a tuberculous mycobacterium. The receptor is raisedto a synthetic immunogen comprising a synthetic polypeptide describedabove alone or as a conjugate.

Also contemplated is a diagnostic system for assaying for the presenceof an antigen to a tuberculous mycobacterium. The system comprisesreceptor molecules as described above and an indicating means forsignaling for the immunoreaction of the combining sites with an antigento a tuberculous mycobacterium.

Further contemplated is a diagnostic system for assaying for thepresence of antibody molecules to an antigen to a tuberculousmycobacterium in a body component. Such a system comprises syntheticpolypeptide as described above and an indicating means for signaling theimmunoreaction of the polypeptide with the antibody molecules to anantigen to a tuberculous mycobacterium. In a more preferred embodiment,this system also contains a solid support comprised of a solid matrix towhich the polypeptide is affixed. A means for identifying the isotype ofthe immunoreacted antibody molecules may also be included in the system.

In another embodiment, the present invention includes a diagnosticsystem for determining the presence of cell-mediated immuneresponsiveness to a tuberculous mycobacterial antigen in a hostcomprising a synthetic polypeptide as described above that has an aminoacid residue sequence that corresponds to the amino acid sequence of atuberculous mycobacterial antigen. The polypeptide, when administered toa host intradermally in an effective amount and in physiologicallytolerable diluent, is capable of inducing the proliferation ofthymus-derived cells in the host. The proliferation is indicated byerythema (redness) and intradermal administration.

Methods are also disclosed for inducing the proliferation ofthymus-derived cells in a host previously immunized to a tuberculousmycobacterium and for determining the presence of a tuberculousmycobacterial antigen in a host. The methods include the steps ofproviding a polypeptide as discussed herein and administeringintradermally an effective amount of the polypeptide to the host in aphysiologically tolerable diluent according to the latter method, theproliferation of thymus-derived cells and the presence of a tuberculousmycobacterial antigen in the host are indicated by erythema andinduration at the site of intradermal administration.

The present invention provides several advantages and benefits. Oneadvantage of the present invention is that use of a syntheticpolypeptide obviates the need for the presence of its correspondingintact protein. The polypeptide itself may provide a vaccine sufficientto protect the host from disease. Consequently, impurities such ascellular debris and toxins that are associated with the production ofusable amounts of viral proteins from bacteria are absent from theproduct of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the 20 amino acid residue sequence of theamino-terminal portion of the BCG-a protein of M. bovis as determined byMinden et al., Infect. Immun., 46, 516 (1984). Synthetic polypeptidescomprising the first 12 amino acid residues of the BCG-a protein arealso illustrated. In the first instance, the synthetic polypeptideincludes cysteine residue at the carboxy-terminus. In the secondinstance, the synthetic polypeptide includes cysteine residues at boththe amino-terminus and the carboxy-terminus.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Humans infected with tuberculous mycobacteria develop antibodies againstantigens associated with the mycobacteria. Traditional clinicaltechniques used to assay for tuberculous mycobacteria andanti-tuberculous mycobacteria antibodies in humans are cumbersome. Inaddition, current procedures for the purification of antigens totuberculous mycobacteria from cell culture are not readily adaptable tomass production.

The present invention contemplates the use of synthetic polypeptidetechnology to overcome some of the problems of the currentmethodologies. Relatively short synthetic polypeptides mayimmunologically mimic antigenic determinants on a natural protein andmay therefore be used to raise antibodies of predetermined specificitythat recognize the natural protein.

The phrase "immunologically mimics" is used herein to mean that animmunogenic polypeptide of this invention induces production ofantibodies that bind to the inducing polypeptide and also to the cognatesequence in the intact protein. This phenomenon may be used bothexperimentally and clinically.

Experimentally, antibodies to synthetic polypeptides may be used toestablish the DNA reading frame, and therefore the amino acid residuesequence of a clinically important protein such as BCG-a protein of M.bovis. Clinically, antibodies of predetermined specificity raised tosynthetic polypeptides may be used for diagnostic and therapeuticpurposes.

Relatively short polypeptides were synthesized whose amino acid residuesequences substantially correspond to that of a tuberculousmycobacterial antigen.

In particular, the invention includes synthethic polypeptides havingamino acid residue sequences that substantially correspond to portionsof the amino acid residue sequence of the BCG-a protein fromMycobacterium bovis strain BCG. This protein was identified by Minden etal., Infect. Immun., 46, 519 (1984), as a protein that is specificallyexpressed by tuberculous mycobacteria (M. bovis and M. tuberculosis). Asreported in the above publication, Minden et al. determined the sequenceof the amino terminal 20 residues of this protein. (See the first aminoacid residue sequence of FIG. 1).

According to the present invention, a polypeptide having an amino acidresidue sequence that corresponds to residues 1-12 of the BCG-a proteinwith a cysteine at the carboxy-terminus was synthesized (see the secondamino acid residue sequence of FIG. 1) and was shown to elicit a delayedcutaneous hypersensitivity reaction to guinea pigs immunized with asonic extract of M. bovis strain BCG.

In addition, in guinea pigs immunized as described above, a morepronounced delayed cutaneous hypersensitivity reaction was elicited by apolypeptide having an amino acid residue sequence that corresponds toresidues 1-12 of the BCG-a protein but includes a cysteine (Cys) residueat both the amino-terminus and the carboxy-terminus. (See the thirdamino acid residue sequence of FIG. 1).

These results indicate that the polypeptides may be utilized in a skintest to detect with specifity infections of the tuberculous mycobacteriain man. The polypeptides may also be used to circumvent thecross-reactivity problems associated with currently used delayed-typecutaneous hypersensitivity antigens. The present polypeptide may replacetuberculin or PPD as the antigen of choice in DCH tests for thedetection of tuberculosis in humans.

A. Synthetic Polypeptides

1. Sequences

The relatively small synthetic polypeptides (13-20 amino acid residuesin length) that were used in this study were synthesized using the solidphase method of Merrifield et. al., J. Am. Chem. Soc., 85, 2149 (1963).

The term "synthetic" as used herein means that the polypeptide moleculeor polypeptide repeating unit has been built up by chemical means; i.e.,chemically synthesized, rather than being prepared by a biologicalmeans, as by genetic engineering techniques. Thus, the syntheticpolypeptides embodying the present invention are free from naturallyoccurring proteins and fragments thereof.

The chemically synthesized polypeptides therefore also differ fromdegradation products of naturally occurring proteins as are prepared bythe action of cyanogen bromide on the protein. The well-know solid phasechemical synthesis in which blocked amino acid residues are added in aserial manner to obtain the desired polypeptide is the preferred methodof synthesis, and is discussed in greater detail hereinbelow.

All amino acid residues identified herein are in the natural ofL-configuration. In keeping with standard polypeptide nomenclature,abbreviations for amino acid residues are as follows:

    ______________________________________                                        SYMBOL                                                                        1-Letter   3-Letter         AMINO ACID                                        ______________________________________                                        Y          Tyr              L-tyrosine                                        G          Gly              L-glycine                                         F          Phe              L-phenylalanine                                   M          Met              L-methionine                                      A          Ala              L-alanine                                         S          Ser              L-serine                                          I          Ile              L-isoleucine                                      L          Leu              L-leucine                                         T          Thr              L-threonine                                       V          Val              L-valine                                          P          Pro              L-proline                                         K          Lys              L-lysine                                          H          His              L-histidine                                       Q          Gln              L-glutamine                                       E          Glu              L-glutamic acid                                   Z          Glx              L-glutamic acid                                                               or L-glutamine                                    W          Trp              L-tryptophan                                      R          Arg              L-arginine                                        D          Asp              L-aspartic acid                                   N          Asn              L-asparagine                                      B          Asx              L-aspartic acid                                                               or L-asparagine                                   C          Cys              L-cysteine                                        ______________________________________                                    

The present invention contemplates a synthetic polypeptide containingabout 13 to about 40 amino acid residues and including the sequencedefined by the formula written from left to right and in the directionof amino-terminus to carboxy-terminus

AlaLysValAsnIleLysProLeuGluAspLysIleCys. The polypeptide is capable,when linked to a carrier and introduced in an effective amount into amammalian host, of inducing production of antibodies that immunoreactwith an antigen to a tuberculous mycobacterium.

The synthetic polypeptides of this invention are often referred toherein simply as "polypeptides" or as "synthetic polypeptides". Thatusage is for brevity.

The term "antigenically related variants" is used herein to designatepolypeptides of differing overall amino acid residue sequence that shareat least a portion of one antigenic determinant and are thereforeimmunologically cross-reactive.

The term "antigenic determinant", as used herein, designates thestructural component of a molecule that is responsible for specificinteraction with corresponding antibody (immunoglobulin) moleculeselicited by the same or related antigen or immunogen.

The term "immunogenic determinant", as used herein, designates thestructural component of a molecule that is responsible for the inductionin a host of an antibody containing an antibody combining site(idiotype) that binds with the immunogen when used as an antigen.

The term "antigen", as used herein, means an entity that is bound by anantibody.

The term "immunogen", as used herein, describes an entity that inducesantibody production in the host animal. In some instances, the antigenand immunogen are the same entity, while in other instances, the twoentities are different.

For example, as is described hereinafter, the polypeptide was used toinduce production of antibodies in a guinea pig and thus, was used as animmunogen. The antibodies so induced bind to the polypeptide when usedas an antigen. The polypeptide was therefore both an immunogen and anantigen. Anti-tuberculous mycobacterium antibodies bind to both thetuberculous mycobacterial antigen as the immunogen and antigen as wellas to the polypeptide as antigen.

Preferred embodiments of the present invention are the syntheticpolypeptides described herein, the pharmaceutically acceptable saltsthereof, and antigenically related variants thereof. Each of thosepolypeptides is capable of inducing antibodies that bind to antigens totuberculous mycobacterium, as described above.

It is noted that a dash at the beginning or end of an amino acid residuesequence indicates a bond to a radical such as H and OH, at the amino-and carboxy-termini, respectively, or a further sequence of one or moreamino acid residues up to a total of forty amino acid residues in thepolypeptide chain.

The phrase "pharmaceutically acceptable salts", as used herein, refersto non-toxic alkali metal, alkaline earth metal and ammonium salts usedin the pharmaceutical industry, including the sodium, potassium,lithium, calcium, magnesium and ammonium salts and the like that areprepared by methods well-known in the art. The phrase also includesnon-toxic acid addition salts that are generally prepared by reactingthe compounds of this invention with a suitable organic or inorganicacid. Representative salts include the hydrochloride, hydrobromide,sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, vorate,benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate,succinate, tartrate, and the like.

B. Multimers

The present invention also contemplates a synthetic multimer containinga plurality of joined synthetic polypeptide repeating units wherein atleast one of the repeating units is a polypeptide as described herein.

The multimers of this invention, alone or linked to a carrier, whenintroduced in an effective amount into a mammalian host, are capable ofinducing the production of antibodies that bind to an antigen to atuberculous mycobacterium. Those multimers that contain the particularlypreferred synthetic polypeptide of this invention are also capable ofbinding human antibodies induced by an antigen to a tuberculousmycobacterium.

Thus, the multimers of this invention, like the polypeptide, areimmunogenic, and are antigenic to human anti-tuberculous mycobacteriumantibodies. Those multimers may therefore be used to induce theproduction of anti-tuberculous mycobacterium antibodies that are usefulin the diagnostic methods and systems discussed hereinfter, and may alsobe used as an antigen in appropriate diagnostic methods and systems.

Multimers that contain fewer than about 35 amino acid residues in thetotal multimer are typically linked to a carrier for use as animmunogen. Those multimers that contain more than a total of about 35amino acid residues are typically sufficiently immunogenic to be usedwithout a carrier.

Polypeptide multimers may be prepared by bonding together thesynthesized polypeptide monomers in a head-to-tail manner using theaforementioned solid phase method; i.e., one complete polypeptidesequence can be synthesized on the resin, followed by one or more of thesame or different polypeptide sequences, with the entire multimeric unitthereafter being cleaved from the resin and used as described herein.Such head-to-tail polypeptide multimers preferably contain about 2 toabout 4 polypeptide repeating units.

Alternatively, multimers can be prepared as a polymer of syntheticpolypeptides used as monomers. As used herein, the term "polymer" in itsvarious grammatical forms is defined as a type of multimer that containsa plurality of synthetic, random copolymer polypeptide repeating unitsthat are joined together by other than peptide bonds.

An exemplary polymer of this invention can be synthesized using apolypeptide of this invention that contains added cysteine residues atboth the amino- and carboxy-termini (diCys polypeptide). The diCyspolypeptide may be bonded together by intramolecular, interpolypeptidecysteine disulfide bonds utilizing an oxidation procedure to form animmunogenic, antigenic polymer. The polymer so prepared contains aplurality of the synthetic polypeptide of this invention as repeatingunits. Those repeating units are bonded together by the above-discussedoxidized cysteine (cystine) residues.

The presence of one or two terminal Cys residues in a polypeptide ofthis invention for the purposes of binding the polypeptide to a carrieror for preparing a polymer is not to be construed as altering the aminoacid sequence of polypeptide repeating units of this invention.

C. Inocula

In another embodiment, the polypeptides of this invention are used in apharmaceutically acceptable diluent to form an inoculum or a vaccinethat, when administered in an effective amount, is capable of inducingantibodies that immunoreact with an antigen to a tuberculousmycobacterium.

The word "inoculum" in its various grammatical forms is used herein todescribe a composition containing a polypeptide of this invention as anactive ingredient used for the preparation of antibodies againsttuberculous mycobacteria. When a polypeptide is used to induceantibodies it is to be understood that the polypeptide may be usedalone, linked to a carrier or as a multimer, but for ease of expression,these alternatives will not alwaysbe expressed hereinafter.

For polypeptides that contain fewer than about 35 amino acid residues,it is preferable to use a carrier for the purpose of inducing theproduction of antibodies. A polypeptide bound or linked to a carrierwill be used illustratively herein where antibodies are being prepared.The inoculum can be used to produce antibodies for use in a diagnosticassays that detect antigens to tuberculous mycobacteria.

The word "vaccine" in its various grammatical forms is used herein todescribe a type of inoculum containing a polypeptide of this inventionas an active ingredient that is used to induce active immunity in a hostmammal. Since active immunity involves the production of antibodies, avaccine or inoculum may thus contain identical ingredients, but theiruses are different. In most cases, the ingredients of a vaccine and ofan inoculum are different because many adjuvants useful in animals maynot be used in humans.

The present inoculum or vaccine contains an effective amount of apolypeptide of this invention, as a multimer such as a polymer ofindividual polypeptides linked together through oxidized, polypeptideterminal cysteine residues or as a conjugate linked to a carrier.However, for ease of expression, the various embodiments of thepolypeptides of this invention are collectively referred to herein bythe term "polypeptide," and its various grammatical forms.

The effective amount of polypeptide per unit dose depends, among otherthings, on the species of animal inoculated, the body weight of theanimal and the chosen inoculation regimen as is well known in the art.Inocula and vaccines typically contain polypeptide concentrations ofabout 100 micrograms to about 500 milligrams per inoculation (dose). Thestated amounts of polypeptide refer to the weight of polypeptide withoutthe weight of a carrier, when a carrier is used. Specific, exemplaryinocula are described hereinafter with weight of carrier pluspolypeptide (conjugate) being given.

The term "unit dose" refers to physically discrete units suitable asunitary dosages for animals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect in association with the required diluent; i.e.,carrier, or vehicle. The specifications for the novel unit dose of thisinvention are dictated by and are directly dependent on (a) the uniquecharacteristics of the active material and the particular therapeuticeffect to be achieved, and (b) the limitations inherent in the art ofcompounding such active material for therapeutic use in animals, asdisclosed in detail in the specification, these being features of thepresent invention.

Inocula are typically prepared from the dried solidpolypeptide-conjugate or polypeptide polymer by suspending thepolypeptide-conjugate or polypeptide polymer in a physiologicallytolerable (acceptable) diluent such as water, saline orphosphate-buffered saline.

Inocula may also include an adjuvant. Adjuvants such as completeFreund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and alum arematerials well known in the art, and are available commercially fromseveral sources.

D. Receptors

Antibodies and substantially whole antibodies raised to (induced by) thepolypeptides of this invention as well as antibody combining sitesprepared from such antibodies constitute still another embodiment ofthis invention. These molecules are collectively referred to herein asreceptors. Receptors are raised in mammalian hosts such as mice, guineapigs, rabbits, horses and the like by immunization using the inoculadescribed hereinabove.

Suitable monoclonal receptors, typically whole antibodies, may also beprepared using hybridoma technology described by Niman et. al., Proc.Natl. Acad. Sci. (U.S.A.), 80, 4949 (1983), which description isincorporated herein by reference. Briefly, to form the hybridoma fromwhich the monoclonal receptor is produced, a myeloma or otherself-perpetuating cell line is fused with lymphocytes obtained from thespleen of a mammal hyperimmunized with a polypeptide of this invention.

It is preferred that the myeloma cell line be from the same species asthe lymphocytes. Typically, a mouse of the strain BALB/c is thepreferred mammal. Suitable mouse myelomas for use in the presentinvention include the hypoxanthine-aminopterin-thymidine-sensitive (HAT)cell lines P3X63-Ag8.653 (ATCC CRL 1580), and Sp2/0-Ag14 (ATCC CRL1581).

Splenocytes are typically fused with myeloma cells using a polyethyleneglycol such as PEG 1500 or PEG 6000. Fused hybrids are selected by theirsensitivity to HAT. Hybridomas producing the receptor molecules of thisinvention are identified using the enzyme linked immunosorbent assay(ELISA) described in the Materials and Methods section hereinafter.

Monocloncal receptors need not only be obtained from hybridomasupernatants, but may also be obtained in generally more concentratedform from ascites fluid of mammals into which the desired hybridoma hasbeen introduced. Production of monoclonal antibodies using ascites fluidis well known and will not be dealt with further herein.

A receptor of this invention binds both to the polypeptide to which itwas raised and also binds to the corresponding tuberculous mycobacteriumantigenic determinant site that the polypeptide of this inventionimmunologically mimics. Thus, a polypeptide of this invention may beboth an immunogen and an antigen.

The receptors of this invention may be described as being oligoclonal ascompared to naturally occurring polyclonal antibodies since they areraised to an immunogen having relatively few epitopes as compared to theepitopes of an intact tuberculous mycobacterium antigenic molecule.Consequently, receptors of this invention bind to epitopes of thepolypeptide while naturally occurring antibodies raised to antigens oftuberculous mycobacteria bind to epitopes throughout the tuberculousmycobacterium antigenic molecule.

E. Diagnostic Assays Systems and Methods

The polypeptides, antibodies and antibody combining sites (receptors)raised to the before described polypeptides, and methods of the presentinvention may also be used for diagnostic tests, such as immunoassays.Such diagnostic techniques include, for example, enzyme immune assay,enzyme multiplied immunoassay technique (EMIT), enzyme-linkedimmunosorbent (ELISA), radio-immune assay (RIA), flourescence immuneassay, either single or double antibody techniques, and other techniquesin which either the receptor or the antigen is labeled with somedetectable tag or indicating means. See generally Maggio, EnzymeImmunoassay, CRC Press, Cleveland, Ohio (1981); and Goldman, M.,Flourescent Antibody Methods, Academic Press, New York, N.Y. (1980).Specific examples of such assay methods and systems useful in carryingout those methods are discussed hereinbelow.

1. Assays For Tuberculous Mycobacteria

A method for assaying for the presence of an antigen to tuberculousmycobacteria in a body sample is also contemplated herein. In a generalmethod, a body sample to be assayed is provided, and is admixed withreceptor molecules that contain an antibody combining site raised to asynthetic polypeptide of this invention. The admixture is maintained fora predetermined period of time sufficient for the receptor molecules toimmunoreact with the antigen to tuberculous mycobacteria present in thebody sample. The amount of that immunoreaction is then measured todetermine whether the tuberculous mycobacterial antigen was present orabsent in the assayed body sample.

An illustrative diagnostic system in kit form embodying one aspect thepresent invention that is useful for detecting tuberculous mycobacterialantigens present in an aliquot of a body sample contains receptormolecules of this invention such as antibodies, substantially wholeantibodies, or antibody combining sites like Fab and F(ab')₂ antibodyportions raised to a polypeptide of this invention in one package. Thissystem also includes an indicating means for signaling the presence ofan immunoreaction between the receptor and the antigen.

Typical indicating means include radioisotopes such as ¹²⁵ I and ¹³¹ I,enzymes such as alkaline phosphatase, horseradish peroxidase,beta-D-galactosidase and glucose oxidase, and fluorochrome dyes such asfluorescein and rhodamine. The indicating means may be linked directlyto receptor of this invention. The indicating means may also be linkedto a separate molecule such as to a second antibody, to an antibodycombining site or to Staphylococcus aureus (S. aureus) protein A thatreacts with (binds to) the receptor of this invention. A specificexample of such a separate molecule indicating means is ¹²⁵ I-labeled S.aureus protein A.

The indicating means permits the immunoreaction product to be detected,and is packages separately from the receptor when not linked directly toa receptor of this invention. When admixed with a body sample such as anacetone-fixed peripheral blood lymphocyte (PBL) smear, the receptormolecule immunoreacts with the tuberculous mycobacterial antigen to forman immunoreactant, and the indicating means present then signals theformation of immunoreaction product.

One embodiment of a diagnostic method for tuberculous mycobacterialantigens is an immunoflourescent assay that includes an amplifyingreagent. In such an assay a PBL smear is acetone-fixed to a plainmicroscope slide. An aliquot of antibodies raised in accordance withthis invention, e.g., raised in rabbits or guinea pigs, generally about100 micrograms to about 500 micrograms, is contacted with the slideusing well-known techniques.

After rinsing away any un-immunoreacted antibodies of this invention,any non-specific binding sites on the slide are typically blocked with aprotein such as bovine serum albumin (BSA), if desired. A second reagent(amplifying reagent) such as complement, or anti-immunoglobulinantibodies, e.g., guinea pig complement, can then be incubated on thetest slide.

After this second incubation, any unreacted of the amplifying reagent isremoved as by rinsing leaving only that which is bound to thefirst-named antibodies on the assay slide. A third reagent (indicatingmeans), e.g., antibody, like goat anti-guinea pig complement, is thenincubated on the test slide. The third reagent is labeled by beinglinked to a flourochrome dye such as fluorescein isothiocyanate (FITC),rhodamine B isothiocyanate (RITC), tetrametylrhodamine isothiocyanate(TRITC), 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), andthe like as are well known in the art.

Any unreacted third reagent is rinsed off after this third incubation,leaving any FITC labeled goat-antiguinea pig complement antibodies thatbind to the complement on the test slide. The presence of the FITClabeled third reagent may be detected using flourescence microscopy andthereby signal the presence of mycobacterial infection.

A preferred diagnostic system, preferably in kit form, useful forcarrying out the above assay method includes, in separate packages, (a)receptors (antibodies) of this invention that immunoreact with atuberculous mycobacterial antigen, (b) a second, amplifying reagent suchas complement, like guinea pig complement, anti-immunogloulin antibodiesor S. aureus protein A that reacts with the receptor, and (c) anindicating means that may be linked directly to the amplifying means ormay be a portion of a separate molecule such as an antibody orantibody-portion that reacts with the amplifying reagent. The indicatingmeans indirectly signals the immunoreaction of the receptor molecule andthe tuberculous mycobacterial antigen through the mediation of theamplifying reagent.

Receptor molecules and separate indicating means of any diagnosticsystem described herein, as well as the above-described amplifyingreagent, may be provided in solution, as a liquid dispersion or as asubstantially dry powder, e.g., in lyophilized form. Where theindicating means is a separate molecule from the amplifying reagent, itis preferred that the indicating means be packaged separately. Where theindicating means is an enzyme, the substrate of the enzyme may also beprovided in a separate package of the system. A solid support such asthe before-described microscope slide, one or more buffers and acetonemay also be included as separately packaged elements in this diagnosticassay system.

The packages discussed herein in relation to diagnostic systems arethose customarily utilized in diagnostic systems. Such packages includeglass and plastic (e.g., polyethylene, polypropylene and polycarbonate)bottles, vials, plastic and plastic-foil laminated envelopes and thelike.

The use of whole, intact, biologically active antibodies is notnecessary in many diagnostic systems such as the immunoflourescent assaydescribed above. Rather, only the immunologically active,idiotype-containing, antigen binding and recognition receptor site;i.e., the antibody combining site, of the antibody molecule may be used.Examples of such antibody combining sites are those known in the art asFab and F(ab')₂ antibody portions that are prepared by proteolysis usingpapain and pepsin, respectively, as is well known in the art.

2. Assays For Anti-Tuberculous Mycobacterial Antibodies

Another diagnostic method of this invention is an ELISA that detectsanti-tuberculous mycobacterial antibodies (such as anti-BGG-aantibodies) in a body sample. Here, a polypeptide of this invention isused as an antigen, and is preferably bound on (adsorbed to) orotherwise linked to a solid matrix such as the cross-linked dextranavailable under the trademark SEPHADEX from Pharmacia Fine Chemicals(Piscataway, N.J.), agarose, beads of glass, polyvinyl chloride,polystyrene, cross-linked acrylamide, nitrocellulose or the wells of amicrotiter plate to form a solid support.

The polypeptide is admixed with a provided body sample to be assayed.The admixture is maintained for a predetermined time sufficient foranti-tuberculous mycobacterial antibodies present in the body sample toimmunoreact with the polypeptide. The presence of that immunoreaction isthen determined as with an indicating means to signal the presence ofanti-tuberculous mycobacterial antibodies in the assayed body sample.

An exemplary ELISA utilizing the above method uses a solid supportcomprised of a polypeptide of this invention adsorbed onto a solidmatrix comprised of the wells of a twelve or ninety-six well microtiterplate made of polystyrene or polyvinyl chloride. Non-specific bindingsites on the microtiter well walls are thereafter typically blocked witha protein such as bovine serum albumin (BSA). Unbound polypeptide andBSA are removed from the microtiter well as by rinsing.

A body sample aliquot such as human serum, blood or plasma is admixedwith the above-described polypeptide-bound solid support to form anadmixture containing solid and liquid phases. The solid-liquid phaseadmixture is maintained for a time sufficient for anti-tuberculousmycobacterial antibodies in the body sample to immunoreact with thepolypeptide antigen. The solid and liquid phases are thereaftergenerally separated.

A solution of a second, labeled, indicating means-containing antibody,antibody combining site or S. aureus protein A that reacts with thefirst-named antibody is then admixed with the solid phase to formanother solid-liquid phase admixture. An exemplary second antibody is aperoxidase-labeled goat anti-human Ig antibody where the first-namedantibodies are from a human body sample. Additional, useful enzymelabels include alkaline phosphase, beta-D-galactosidase and glucoseoxidase. The admixture formed from the solid phase and the secondlabeled antibody solution is maintained (incubated) for a predeterminedtime period (e.g., 30 minutes) sufficient to form a reactant between thefirst-named antibody and the indicating means such as an immunoreactionbetween the two antibodies. The solid and liquid phases are thereafterseparated.

The second antibody described above may also be specific for andimmunoreact with only one of the classes of immunoglobulin (e.g., IgG,IgM, IgE, IgA, or IgD). Such antibodies may provide the ability toidentify the immunoglobulin class of anti-tuberculous mycobacterialantibody present in the body sample. In addition, the second antibody orantibody combining site may be specific for and immunoreact with onlyone of the two types of immunoglobulin light chains (e.g., kappa orlambda). These antibodies may provide the ability to identify theisotype of the immunoglobulin molecule present in the body sample.

A solution containing a substrate for the enzyme label such as hydrogenperoxide for peroxidase and a color-forming dye precursor such aso-phenylenediamine, or p-nitrophenyl phosphate for alkaline phosphataseis thereafter admixed with the solid phase. The optical density at apreselected wavelength (e.g., 490 or 405 nanometers, respectively) maythen be determined after a predetermined time period has elapsed (e.g.,60 minutes), and compared to the optical density of a control todetermine whether anti-tuberculous mycobacterial antibodies were presentin the body sample.

Another embodiment of this invention comprises a diagnostic system inkit form that includes a solid support comprised of a solid matrix suchas a polystyrene twelve-well microtiter strip, and a polypeptide of thisinvention, absorbed (bound) or otherwise affixed to the solid matrix toform a solid matrix. This system preferably also includes separatelypackaged anti-human Ig antibodies having a linked indicating means suchas peroxidase-labeled goat anti-human Ig antibodies, and may alsoinclude substrate for the linked indicating means such as hydrogenperoxide and a color forming due precursor such as o-phenylenediamine,in further, separate packages. Hydrogen peroxide is typically notincluded in the kit due to its relative instability, and is typicallysupplied by the end user. Buffer salts useful in an assay utilizing thissystem may also be included in one or more separate packages in dry orliquid form. Separate packages containing human anti-tuberculousmycobacterial antibodies and human antibodies free from anti-tuberculousmycobacterial antibodies (normal human antibodies) may also be includedas positive and negative controls, respectively. An assay for thepresence of anti-tuberculous mycobacterial antibodies in a body samplesuch as serum may be carried out with this diagnostic system using theabove-described method.

II. METHODS AND MATERIALS

A. Synthesis of Polypeptides

The polypeptides of this invention were chemically synthesized bysolid-phase methods as described in Merrifield et. al., J. Am. Chem.Soc., 85, 2149 (1963) and Houghten et. al., Int. J. Pept. Prot. Res.,16, 311 (1980). The solid phase method of polypeptide synthesis waspracticed utilizing a Beckman Model 990B Polypeptide Synthesizer,available commercially from Beckman Instrument Co., Berkeley, CA.

For polypeptides having fewer than 35 residues that were used ininocula, a cysteine residue was added to the carboxy-terminus or to boththe amino-terminus and the carboxyl-terminus to assist in coupling to aprotein carrier as described below. The compositions of all polypeptideswere confirmed by amino acid analysis.

In preparing a synthetic polypeptide of this invention by the abovesolid phase method, the amino acid residues are linked to a resin (solidphase) through an ester linkage from the carboxy-terminal residue. Whenthe polypeptide is to be linked to a carrier via a Cys residue orpolymerized via terminal Cys residues, it is convenient to utilize thatCys residue as the carboxy-terminal residue that is ester-bonded to theresin.

The alpha-amino group of each added amino acid is typically protected bya tertiary-butoxycarbonyl (t-BOC) group prior to the amino acid beingadded into the growing polypeptide chain. The t-BOC group is thenremoved prior to addition of the next amino acid to the growingpolypeptide chain.

Reactive amino acid side chains were also protected during synthesis ofthe polypeptides. Usual side-chain protecting groups were used for theremaining amino acid residues as follows: O-(p-bromobenzyloxycarbonyl)for tyrosine; O-benzyl for threonine, serine, aspartic acid and glutamicacid; S-methoxybenzyl for cysteine, dinitrophenyl for histidine;2-chlorobenzoxycarbonyl for lysine and tosyl for arginine.

Protected amino acids were recrystallized from appropriate solvents togive single spots by thin layer chromatography. Couplings were typicallycarried out using a ten-fold molar excess of both protected amino acidand dicyclohexyl carbodiimide over the number of milliequivalents ofinitial N-terminal amino acid. A two molar excess of both reagents mayalso be used. For asparagine, an equal molar amount ofN-hydroxy-benzotriazole was added to the protected amino acid anddimethyl formamide was used as the solvent. All coupling reactions weremore than 99% complete by the picric acid test of Gisin, Anal. Chem.Acta., 58, 248 (1972).

After preparation of a desired polypeptide, a portion of the resulting,protected polypeptide (about 1 gram) was treated with two milliliters ofanisole, and anhydrous hydrogen flouride, about 20 milliliters, wascondensed into the reaction vessel at dry ice temperature. The resultingmixture was stirred at about 4 degrees C. for about one hour to cleavethe protecting groups and to remove the polypeptide from the resin.After evaporating the hydrogen flouride at a temperature of 4 degrees C.with a stream of N₂, the residue was extracted with anhydrous diethylether three times to remove the anisole, and the residue was dried invacuo.

The vacuum dried material was extracted with 5 percent aqueous aceticacid (3 times with 50 milliliters) to separate the free polypeptide fromthe resin. The extract-containing solution was lyophilized to provide amonomeric unoxidized polypeptide.

The produced synthetic polypeptide may be used as a reagent in anenzyme-linked immunosorbent assay (ELISA) to detect anti-tuberculousmycobacterial antibodies. The synthetic polypeptide may also be used toproduce an inoculum, usually by linking it to a carrier to formconjugate and then dispersing an effective amount of the conjugate in aphysiologically tolerable diluent, as is discussed hereinafter.

It is also to be noted that a synthetic multimer of this invention canbe prepared by the solid phase synthesis of a plurality of thepolypeptides of this invention linked together end-to-end (head-to-tail)by an amide bond between the carboxyl-terminal residue of onepolypeptide and the amino-terminal residue of a second polypeptide. Suchsynthetic multimers are preferably synthesized as a single longpolypeptide multimer, but can also be prepared as individualpolypeptides that are linked together subsequent to their individualsyntheses, using a carbodiimide reagent such as1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride in water.The total number of amino acid residues contained in a multimer preparedas a single polypeptide chain is preferably less than about 50, so thatup to about eight polypeptides of this invention can be incorporatedinto a single head-to-tail multimer chain that is synthesized as asingle polypeptide. A synthetic head-to-tail multimer more preferablycontains two to about four blocks of linked, synthetic, random copolymerpolypeptides of this invention, and a total of less than about 40 aminoacid residues.

B. Preparation of Polymers

The polypeptides of the present invention may be connected together toform an antigenic and/or immunogenic polymer (synthetic multimer)comprising a plurality of the polypeptide repeating units. Such apolymer typically has the advantage of increased immunogenicity andantigenicity. In addition, a carrier is typically not needed when apolymeric immunogen is utilized. Where different polypeptide monomersare used to make up the polymer, the ability to immunoreact withantibodies to several tuberculous mycobacterial antigenic determinantsis obtained. A still further advantage is the ability of such a polymerwhen used in an inoculum to induce antibodies that immunoreact withseveral antigenic determinants of a tuberculous mycobacterial antigen.

A polymer of this invention may be prepared by synthesizing thepolypeptides as discussed above and including cysteine residues at boththe amino- and carboxy-termini to form a "diCys-terminated" polypeptide.After synthesis, in a typical laboratory preparation, 10 milligrams ofthe diCys polypeptide (containing cysteine residues in un-oxidized form)are dissolved in 250 milliliters (ml) of 0.1 molar (M) ammoniumbicarbonate buffer. The dissolved diCys-terminated polypeptide is thenair oxidized by stirring the resulting solution gently for a period ofabout 18 hours in the air, or until there is no detectable freemercaptan by the Ellman test. [See Ellman, Arch. Biochem. Biophys., 82,70 (1959).]

The polymer (synthetic multimer) so prepared contains a plurality of thesynthetic, random copolymer polypeptide repeating units that are bondedtogether by oxidizing cysteine (cystine) residues. Such polymerstypically contain their polypeptide repeating units bonded together in ahead-to-tail manner as well as in head-to-head and tail-to-tail manners;i.e., the amino-termini of two polypeptide repeating units may be bondedtogether through a single cystine residue as may two carboxyl-terminisince the linking groups at both polypeptide termini are identical.

C. Coupling To Carriers

The synthetic polypeptides were coupled to keyhole limpet hemocyanin(KLH) as carrier by the method described in Liu et al., Biochem., 80,690 (1979). Briefly, 4 milligrams (mg) of the carrier were activatedwith 0.51 mg of m-maleimidobenzoyl-N-hydroxysuccinimide ester, and weresubsequently reacted with 5 mg of the polypeptide through an amino- orcarboxy-terminal cysteine to provide a conjugate containing about 10 toabout 35 percent by weight polypeptide.

One or more additional amino acid residues may be added to the amino- orcarboxy- termini of the synthetic polypeptide to assist in binding thepolypeptide to a carrier. As discussed before, cysteine residues addedat the amino- or carboxy-termini of the synthetic polypeptide have beenfound to be particularly useful for forming polymers via disulfidebonds. However, other methods well known in the art for preparingconjugates can also be used. Exemplary additional linking proceduresinclude the use of Michael addition reaction products, dialdehydes suchas glutaraldehyde, Klipstein et al., J.Infect. Dis., 147, 318 (1983) andthe like, or the use of carbodiimide technology as in the use of awater-soluble carbodiimide to form amide links to the carrier, asdiscussed before for linking a plurality of polypeptides together toform a synthetic multimer.

Useful carriers are well known in the art, and are generally proteinsthemselves. Exemplary of such carriers are keyhole hemocyannn (KLH),edestin, thyroglobulin, albumins such as bovine serum albumin (BSA) orhuman serum albumin (HSA), red blood cells such as sheep erthrocytes(SRBC), tetanus toxoid, cholera toxoid as well as polyamino acids suchas poly (D-lysine:D-glutamic acid), and the like.

As is also well known in the art, it is often benefical to bind asynthetic polypeptide to its carrier by means of an intermediate,linking group. As noted above, glutaraldehyde is one such linking group.However, when cysteine is used, the intermediate linking group ispreferably an m-maleimidobenxoyl N-hydroxy succinimide (MBS), as wasused herein.

Additionally, MBS may be first added to the carrier by an ester-amideinterchange reaction as disclosed by Liu et al., supra. Thereafter, theaddition can be followed by addition of a blocked mercapto group such asthiolacetic acid (CH₃ COSH) across the maleimido-double bond. Aftercleavage of the acyl blocking group, a disulfide bond is formed betweenthe deblocked linking group mercaptan and the mercaptan of the addedcysteine residue of the synthetic polypeptide.

The choice of carrier is more dependent upon the ultimate use of theimmunogen than upon the determinant portion of the immunogen, and isbased upon criteria not particularly involved in the present invention.For example, if a inoculum is to be used in animals, a carrier that doesnot generate an untoward reaction in the particular animal should beselected.

D. ELISA

Anti-polypeptide antibody binding and inhibition studies may be preparedby an enzyme-linked immunosorbent assay (ELISA) as described below.

Briefly, microtiter wells (Costar, #3590, Cambridge, MA) are coated withindividual polypeptides as antigens by adding 100 microliters (ul) ofBBS [10 millimoler (mM) sodium borate (pH 8.3), 150 mM NaCl] containingpolypeptide at a concentration of 10 micrograms per milliliter (ug/ml).Contact between the wells and antigen-containing solution is maintainedfor a predetermined time, typically 15 minutes, and at 20 degrees C., toform an antigen-coated solid phase. The solid and liquid phases areseparated and the wells are washed three times with BBS.

Non-specific binding sites are blocked by admixing 200 microliters of 1percent bovine serum albumin (BSA) in each well to form anothersolid-liquid phase admixture, and maintaining that solid-liquid phaseadmixture for 30 minutes, at 20 degrees C. The phases are separated andexcess, unbound BSA is removed by washing three times with BBS.

Rabbit (or guinea pig) and human sera (body sample aliquots) are assayedfor anti-polypeptide activity by adding 100 microliters of a serumdiluted 1:20 in BBS per well to form a solid/liquid phase composition.Contact between the diluted sera and the antigen-coated solid phase ismaintained for a predetermined time such as 1 hour, and at 20 degreesC., for an immunoreactant to form. The solid and liquid phases areseparated, and the solid phase; i.e., antigen-coated,immunoreactant-containing wells, is then washed three times with BBS.

The antibodies in human sera that immunoreact with an adsorbedpolypeptide may be detected using an indicating means comprisingalkaline phosphatase-conjugated goat anti-human Ig antibody (Tago,Burlington, CA). The antibodies in rabbit sera that immunoreact with anadsorbed polypeptide may be detected using an indicating meanscomprising alkaline phosphatase-conjugated goat anti-rabbit Ig antibody(Kirkegard & Perry Laboratories, Inc., Gaithersburg, MD). In eitherinstance, 100 microliters of the indicating antibody diluted 1:300 inBBS are added per well to form a further solid-liquid phase composition.This solid-liquid phase composition is maintained for a predeterminedtime, one hour, for the formation of a reaction product between thehuman antibodies bound to the solid phase and the indicating means, andat 20 degrees C. The phases are separated, and the solid phase is washed3 times with BBS.

Alkaline phosphatase-conjugated antibody bound to polypeptide specificantibody may be detected by spectrophotometrically measuring theenzymatic hydrolysis of p-nitrophenyl phosphate to p-nitrophenol.Briefly, 100 microliters of p-nitrophenyl phosphate [1 milligram permilliliter in 2 mM magnesium chloride (pH 9.8), 50 mM sodium carbonate]are added to each well. The enzymatic reaction is allowed to proceed 1hour and then the optical density at 405 nm is determined in a TITERTEKspectrophotometer available from Flow Laboratories, Inglewood, CA.

E. Immunizations

The receptor molecules of this invention include whole antibodies raisedin mammals by immunizing them with inocula including a polypeptideand/or multimer as described hereinabove. Both polypeptides andmultimers may be used included in inocula alone or conjugated to acarrier protein such as keyhole limpet hemocyamin (KLH). However,polypeptides are preferably used as a conjugate and multimers arepreferably used alone.

Rabbits may be immunized with inocula containing 1.0 mg of conjugate incomplete Freund's adjuvant (CFA), and boosted one month later with 1.0mg of conjugate in incomplete Freund's adjuvant (IFA). Each immunizationconsisted of one subcutaneous injection, on the back hip. Rabbits werebled 1 and 2 months subsequent to the boost.

Sera containing immunologically active antibodies were then producedfrom the bleeds by methods well known in the art. These antibodiesimmunoreacted with one or more of the polypeptides of this invention,and a a tuberculous mycobacterial antigenic determinant. They may thusbe used in a system to determining the presence of mycobacterialinfections.

Individual inocula are prepared with CFA or IFA as follows: An amount ofconjugate sufficient to provide the desired amount of polypeptide perinoculation (e.g., 1 mg) is dissolved in PBS (at about 0.5 ml) at pH7.2. Equal volumes of CFA or IFA are then mixed with the conjugatesolutions to provide an inoculum containing conjugate, water andadjuvant in which the water to oil ratio was 1:1. The mixture isthereafter homogenized to provide the inocula. The volume of an inoculumso prepared is typically greater than 1 ml, and some of the conjugate,PBS and adjuvant may be lost during the emulsification. Substantiallyall of the emulsion that can be recovered is placed into a syringe, andthen is introduced into the rabbits as discussed before. The amount ofinoculum introduced into the rabbits should be at least about 90 percentof that present prior to the emulsification step.

The above inocula stock solutions are illustrative of the inocula ofthis invention. As demonstrated herein, they may be used to producereceptor molecules that immunoreact with tuberculous mycobacterialantigens.

F. Delayed-Type Hypersensitivty Reaction (Skin Reaction Test)

The previously described diagnostic systems and assays are based on invitro assays. Although particular steps of the assays can be carried outin vivo, the actual immune response is measured in tissue culture. Thepresent invention, however, can also be applied to diagnostic systemsinvolving the in vivo measurement of T cell responses. One example ofsuch a system is a delayed-type cutaneous hypersensitivity (DCH)reaction or what is more commonly known as a skin reaction test.

A DCH reaction can only occur in an individual previously exposed(sensitized) to a given antigen. The first exposure of an individual tothe antigen produces no visible change, but the immune status of theindividual is altered in that hypersensitivity to renewed exposure tothat antigen results. Thus, upon intradermal or subcutaneous injectionof the antigen (preferably in a buffered saline solution) acharacteristic skin lesion develops at the injection site - a lesionthat would not develop after a first antigen exposure. Because theresponse to the second (or challenge) antigen inoculum is typlcallydelayed by 24 to 48 hours, the reaction is referred to as delayed-typehypersensitivity.

In humans, exposure to a sensitizing antigen takes place upon contactwith the microorganism responsible for the disease (e.g., tuberculinfrom Mycobacterium tuberculosis, typhoidin from Salmonella typhi andabortin from Brucella abortus), and sensitization occurs as a result ofa chronic infection. In animals, sensitization can be achieved byinoculation of an antigen emulsified in water, saline or an adjuvant.

In both humans and animals, hypersensitivity is tested in vivo by theinjection of the antigen dissolved in a physiologically tolerablediluent such as saline solution into the skin (either intradermally orsubcutaneously). DCH is usually a more sensitive diagnostic assay thanthe determination or measurement of the amount of antibody produced toan antigen. For example, only minute amounts of protein (a few hundredmicrograms) are necessary for DCH sensitization of a mouse, while a muchlarger dose is needed to induce antibody production.

Since the polypeptides of the present invention stimulate theproliferation of guinea pig T cells following immunization(sensitization) with a polypeptide of the invention, a skin reactiontest was developed using one or more of the present syntheticpolypeptides as a challenge antigen.

In particular, a delayed-type cutaneous hypersensitivity reaction wasobserved when a synthethic polypeptide of this invention wasadministered intradermally to Mycobacterium bovis BCG-sensitized andMycobacterium tuberculosis strain H37Rv-sensitized guinea pigs.

The bacteria described herein were obtained from the culture collectionof the Scripps Clinic and Research Foundation and were grown asdescribed in Minden et al., Science, 176, 57 (1972) and Minden et al.,Infect. Immun., 6,574 (1972), which publications are incorporated hereinby reference.

In particular, guinea pigs that were immunized about six weeks earlierwith heat-killed BCG-containing or H37Rv-containing cell sonicatesdemonstrated a delayed cutaneous hypersensitivity (DCH) reaction uponintradermal infection of about 250 micrograms of a syntheticpolypeptide. The polypeptide was typically administered in about 100microliters of phosphate-buffered saline (pH 7.0) containing 0.005percent TWEEN 20.

In conventional practice, a DCH reaction is sought at 24 and 48 hoursafter injection of an antigen. The inflammatory infiltrate (consistingprimarily of mononuclear cells), which occurs 24-48 hours followingintradermal injection of the antigen, and the accompanying edema produceinduration of the skin. A coexistent area of erythema may also develop.The diameter of this induration is an index of cutaneoushypersensitivity, and an induration of about 5 millimeters or more indiameter is the generally accepted criterion of a positive DCH reaction.

Polypeptides of the invention elicited an erythematous area and aninduration of at least about 10 millimeters in diameter about theinjection site. Unimmunized animals demonstrated no DCH reaction uponintradermal injection of a polypeptide. Animals immunized with cellextracts of nontuberculous mycobacteria demonstrated marginal DCHreactions (erythemas and indurations of less than about 5 millimeters)to a polypeptide.

The polypeptide that elicited the most pronounced DCH reactions waspolymerized through cysteine residues at both its amino-terminus andcarboxy-terminus. See the di-Cys polypeptide shown at line 3 of Fig. 1.The results of DCH tests using that polypeptide are shown in Table 1.

                  TABLE I                                                         ______________________________________                                                        DCH.sup.2 Reaction To 250                                                                     DCH                                           Animals Immunized With                                                                        Micrograms Of   Reaction                                      Sonicates.sup.1 Of The Following                                                              Polypeptide.sup.3                                                                             To PPD.sup.5                                  ______________________________________                                        M. Bovis strain BCG                                                                           7/7.sup.4       7/7                                           M. tuberculosis strain H37Rv                                                                  4/4             4/4                                           M. fortuitum    0/4             4/4                                           M. kansasii     0/3             3/3                                           M. intracellulare                                                                             2/4             4/4                                           Polypeptide (diCys Terminated)                                                                5/5             0/5                                           (see Note 3)                                                                  Unimmunized Animals                                                                           0/5             0/5                                           ______________________________________                                         .sup.1 Prepared and immunized as described in Minden et al., Infect.          Immun., 46, 519 (1984), which is incorporated herein by reference.            .sup.2 Delayed-type cutaneous hypersensitivity.                               .sup.3 250 micrograms of a polypeptide having the amino acid residue          sequence  CysAlaLysValAsnIleLysProLeuGlnAspLysIleCys (The polypeptide         shown at line 3 of FIG. 1) were dissolved in about 100 microliters of         phosphatebuffered saline (pH 7.0) containing about 0.005 percent TWEEN 20     (ICI Americas, Inc., Wilmington, D).                                          .sup.4 The first numeral in each series represents the number of positive     DCH reactions and the second numeral represents the total number of           trials.                                                                       .sup.5 PPD (Purified Protein Derivative) from Connaught Laboratories LTD.     Willowdale, Canada was administered in 100 microliter quantities which is     equivalent to about 250 tuberculin units.                                

Referring to Table I, guinea pigs immunized with cell sonicates of M.bovis strained BCG or M. tuberculosis H37Rv strain developed a strongDCH reaction to the polypeptides and to PPD (purified proteinderivative).

Animals immunized with nontuberculous mycobacteria (M. fortuitum, M.kansasii and M. intracellulare) developed a weak reaction or did notreact (See Table I for particulars) to the polypeptides, but developed astrong DCH reaction to PPD.

Animals immunized with the diCys-terminated polypeptide developed a DCHreaction to the polypeptide, but not to PPD.

Unimmunized normal animals developed no DCH reaction to the polypeptidesor to PPD.

The immunogenicity of the present polypeptides was also investigatedusing the polypeptides to elicit antibodies. The specificities andbinding characteristics were determined of the resulting antibodies inan enzyme-linked immunosorbent assay (ELISA) as described herein.

A strong anti-polypeptide response was elicited when the polypeptide(shown at line 2 of FIG. 1) was coupled to a protein carrier (keyholelimpet hemocyanin) and injected into rabbits along with incompleteFreund's adjuvant. The titer of the resulting antisera against thepolypeptide was measured by immobilizing 1 microgram of the polypeptidein each well of a polystyrene microtiter plate and then reacting thebound polypeptide with serial dilutions of the antisera. Antibody titeris expressed as the reciprocal of the dilution of antibody that produces50 percent maximal binding in the ELISA. The antisera hadanti-polypeptide titers of 1250-2500.

The reactivity of the polypeptide-elicited antibodies with extracts ofvarious mycobacterial species was measured in an ELISA in which "Santigen" preparations were immobilized in the wells of the polystyrenemicrotiter plates. ("S antigens" are the antigens present in thesupernatant fractions following centrifugation at 100,000xg of bacterialsuspensions that had been disrupted by sonication. As described byMinden et al., Infect. Immun., 46, 519 (1984), which is incorporatedherein by reference).

In the case of the polypeptide having an amino acid residue sequencethat corresponds to residues 1-12 of the BCG-a protein with a cysteineat the carboxy-terminus, the anti-polypeptide antibodies reactedstrongly with the polypeptide (titer=1250), purified BCG-a protein(1250), and soluble sonicated extracts of Mycobacterium bovis strain BCG(675) and Mycobacterium tuberculosis strain H37Rv (675). Only marginalbinding to the sonicated extracts of Mycobacterium fortuitum (50),Mycobacterium kansasii (25) and Mycobacterium intracellulare (25) wasobserved. This sort of maginal binding was also seen with the pre-immunerabbit serum, suggesting that the rabbit antibodies might benon-specifically sticking to these mycobacterial extracts. There was nobinding (titer less ten 10) to the "S antigens" of E. coli, Listeriamonocyogenes, Salmonella epidermis, Salmonella typhimurium, orPseudamonas sp. These data indicate that the polypeptide contains animmunogenic epitope that is expressed predominantly in the tuberculousmycobacterial species.

The antigenicity of the polypeptides was determined in two ways. First,to measure the reactivity with humoral antibodies, the polypeptides wereimmobilized onto wells of a microtiter plate and reacted with variousantibody preparations. In the case of the polypeptide having an aminoacid residue sequence that corresponds to residues 1-12 of the BCG-aprotein with a cysteine at the carboxy-terminus, there was significantbinding to the polypeptide by antibodies directed against thepolypeptide (titer=1250), BCG-a protein (625), and a whole sonicateextract of M. bovis BCG (625). There was only marginal binding by theantibodies elicited by extracts of M. kansaii (25) and M. fortuitum (5).There was no detectable binding to antisera elicited by extracts of E.coli, L. monocytogenes, S. epidermis, S. typhimurium, or Pseudamonas sp.Once again, the data suggest that this epitope is expressed mainly bythe tuberculous species and poorly if at all by the two atypical strainstested. (M. kansasii and M. fortuitum).

The results of the DCH reactions demonstrate that the syntheticpolypeptides of the present invention may be useful in an in vivodiagnostic system for the presence of a cell mediated immune response totuberculous mycobacterial antigens.

After the safety and effectiveness of the above polypeptides are shownin animal studies, the polypeptides can be used as challenge antigens inhuman skin reaction tests for recipients of tuberculous mycobacterialvaccines. The polypeptides are synthesized as previously described,purified by high pressure liquid chromatography (HPLC) techniques,sterilized and pyrogen-tested.

Since the T cell proliferative responses of human tuberculousmycobacterial vaccine recipients can be quite variable relative topolypeptide specificity, vaccine recipients and individuals serving asunvaccinated controls are challenged with a series of polypeptides. Thekinetics and optimal antigen dose can be determined in the vaccinerecipient group using the results from the animal studies as aguideline.

Chronically infected individuals can also be studied for tuberculousmycobacterial-specific T cell sensitization using synthetic polypeptidesas antigens for a skin reaction test.

In each instance, the challenge antigen is administered by intradermalinjection of the particular polypeptide in a physiologically acceptablesolution (about 1 milliliter) into the volar surface of the forearm. Useof a 25- or 27-gauge needle usually assures intradermal rather thansubcutaneous administration of the antigen. Subcutaneous injection canlead to dilution of the antigen in tissues and can produce afalse-negative test. The injection sites are then observed for erythema(skin reddening) and induration (swelling) at 4, 24 and 48 hourspost-challenge.

The foregoing is intended as illustrative of the present invention butnot limiting. Numerous variations and modifications may be effectedwithout departing from the true spirit and scope of the novel conceptsof the invention. It is to be understood that no limitation with respectto the specific polypeptides, antibodies, their compositions and usesillustrated herein is intended or should be inferred.

What is claimed is:
 1. A receptor molecule containing an antibodycombining site raised to a synthetic immunogen, the synthetic immunogencomprising a synthetic polypeptide containing about 13 to about 40 aminoacid residues and including the thirteen amino acid residue sequence,written from left to right in the direction of the amino-terminus tocarboxy-terminus, represented by the formula:

    AlaLysValAsnIleLysProLeuGluAspLysIleCys;

said receptor immunoreacting with an antigen to a tuberculousmycobacterium.
 2. A diagnostic system in kit form for assaying for thepresence of tuberculous mycobacterium comprising:(a) receptor moleculesraised to a synthetic polypeptide.containing about 13 to about 40 aminoacid residues and including the thirteen amino acid residue sequence,written from left to right in the direction of the amino-terminus tocarboxy-terminus, represented by the formula:

    AlaLysValAsnIleLysProLeuGluAspLysIleCys; and

(b) indicating means for signalling the immunoreaction of the receptorwith an antigen to a tuberculous mycobacterium.
 3. The diagnostic systemof claim 2 wherein whole antibodies are said receptor molecules.
 4. Thediagnostic system of claim 2 wherein the receptor molecules are providedin solution or in liquid form in a separate package from said indicatingmeans.
 5. The diagnostic system of claim 2 wherein the receptormolecules are provided in lyophilized form in a separate package.
 6. Thediagnostic system of claim 2 wherein said receptor molecules are labeledwith an indicating means.
 7. The diagnostic system of claim 2 whereinsaid indicating means is a labeled antibody that immunoreacts with saidamplifying reagent.
 8. The diagnostic system of claim 7 wherein saidlabeled antibody is labeled with a fluorochrome dye.
 9. The diagnosticsystem of claim 7 wherein said labeled antibody is labeled withfluorescence isothiocyanate.
 10. The diagnostic system of claim 7wherein said labeled antibody is labeled with an enzyme.
 11. A method ofassaying for antituberculous mycobacterium antibodies in a body samplecomprising the steps of:(a) providing a body sample to be assayed; (b)admixing said body sample with a synthetic polypeptide containing about13 to about 40 amino acid residues and including the thirteen amino acidresidue sequence, written from left to right in the direction of theamino-terminus to carboxy-terminus, represented by the formula:

    AlaLysValAsnIleLysProLeuGluAspLysIleCys;

(c) maintaining said admixture for a predetermined time sufficient foranti-tuberculous mycobacterium antibodies present in said sample toimmunoreact with said polypeptide; and (d) determining the presence ofsaid immunoreaction.
 12. The method of claim 11 wherein said body sampleis selected from the group consisting of blood, serum and plasma. 13.The method of claim 11 including the further step of affixing saidpolypeptide to a solid support prior to said admixing.
 14. A diagnosticsystem in kit form for assaying for the presence of antibodies to anantigen of a tuberculous mycobacterium in a body component comprising inseparate packages:(a) a synthetic polypeptide containing about 13 toabout 40 amino acid residues and including the thirteen amino acidresidue sequence, written from left to right in the direction of theamino-terminus to carboxy-terminus, represented by the formula:

    AlaLysValAsnIleLysProLeuGluAspLysIleCys; and

(b) an indicating means for signaling the immunoreaction of saidpolypeptide with antibodies to the antigen of the tuberculousmycobacterium.
 15. The diagnostic system of claim 14 further including asolid matrix capable of binding said polypeptide.
 16. The diagnosticsystem of claim 15 wherein said synthetic polypeptide is affixed to saidsolid matrix to form a solid support.
 17. The diagnostic system of theclaim 15 wherein said solid matrix is selected from the group consistingof polystyrene, polyvinylchloride and nitrocellulose.
 18. The diagnosticsystem of claim 15 wherein said solid matrix is a microtiter stripcontaining a plurality of wells.
 19. The diagnostic system of claim 14wherein said indicating means is labeled antibody capable ofimmunoreacting with human anti-tuberculous mycobacterium antibodies. 20.The diagnostic system of claim 19 wherein said labeled antibody islabeled with an enzyme selected from the group of enzymes consisting ofalkaline phosphatase, horseradish perodidase, beta-D-galactosidase andglucose oxidase.
 21. A method of assaying for the presence of an antigento a tuberculous mycobacterium in a body sample comprising the stepsof:(a) providing a body sample to be assayed; (b) admixing receptormolecules containing an antibody combining site raised to a syntheticpolypeptide having about 13 to about 40 amino acid residues andincluding the thirteen amino acid residue sequence, written from left toright in the direction of the amino-terminus to carboxy-terminus,represented by the formula:

    AlaLysValAsnIleLysProLeuGluAspLysIleCys;

said polypeptide when linked to a carrier and introduced in an effectiveamount into a mammalian host being capable of inducing the production ofantibodies that immunoreact with an antigen to a tuberculousmycobacterium present in said body sample; and (d) measuring the amountof said immunoreaction.
 22. The method of claim 21 wherein said bodysample is selected from the group consisting of lymphocytes and tumortissue.
 23. An immunoreaction product comprising a humananti-tuberculous mycobacterium antibody immunologically bound to asynthetic polypeptide containing about 13 to about 40 amino acidresidues and including the thirteen amino acid residue sequence, writtenfrom left to right and in the direction of the amino-terminus tocarboxy-terminus, represented by the formula:

    AlaLysValAsnIleLysProLeuGluAspLysIleCys.


24. A diagnostic system for determining the presence of a tuberculousmycobacterial antigen in a host comprising a package containing at leastone synthetic polypeptide having an amino acid residue sequence takenfrom left to right and in the direction from amino-terminus tocarboxy-terminus selected from the group consisting of:

    AlaLysValAsnIleLysProLeuGluAspLysIleCys; and

    CysAlaLysValAsnIleLysProLeuGluAspLysIleCys;

said synthetic polypeptide, when administered intradermally in aphysiologically tolerable diluent to the host, being capable of inducingthe proliferation of thymus-derived cells in the host, saidproliferation being indicated by erythema and induration at the site ofintradermal administration.
 25. The diagnostic system according to claim24 wherein said physiologically tolerable diluent is a member of thegroup consisting of water, saline and an adjuvant.
 26. The diagnosticsystem according to claim 24 wherein said synthetic polypeptide is boundto a carrier.
 27. The diagnostic system according to claim 26 whereinsaid carrier is selected from the group consisting of keyhole limpethemocyanin, keyhole limpet hemocyanin in incomplete Freund's adjuvant,alum, keyhole limpet hemocyanin-alum absorbed, keyhole limpethemocyanin-alum absorbed-pertussis, edestin, thyroglobulin, tetanustoxoid, tetanus toxoid in incomplete Freund's adjuvant, cholera toxoidand cholera toxoid in incomplete Freund's adjuvant.
 28. A method forinducing the proliferation of thymus-derived cells in a host previouslyimmunized to a tuberculous mycobacterial antigen comprising:(a)providing a synthetic polypeptide having an amino acid residue sequencetaken from left to right and in the direction from amino-terminus tocarboxy-terminus selected from the group consisting of:

    AlaLysValAsnIleLysProLeuGluAspLysIleCys; and

    CyAlaLysValAsnIleLysProLeuGluAspLysIleCys;

administering an effective amount of said synthetic polypeptide to thehost in a physiologically tolerable diluent.
 29. The method according toclaim 28 including the step after step (a) but before step (b) oflinking said synthetic polypeptide to a carrier to form a conjugate. 30.A method for determining the presence of a tuberculous mycobacterialantigen in a host comprising:(a) providing a synthetic polypeptidehaving an amino acid residue sequence taken from left to right and inthe direction from amino-terminus to carboxy-terminus selected from thegroup consisting of:

    AlaLysValAsnIleLysProLeuGluAspLysIleCys; and

    CysAlaLysValAsnIleLysProLeuGluAspLysIleCys;

(b) administering intradermally an effective amount of said syntheticpolypeptide dissolved or dispersed in a physiologically tolerablediluent to induce the proliferation of thymus-derived cells in thehostwhereby said proliferation and the presence of a tuberculousmycobacterial antigen in a host is indicated by erythma and indurationat the site of intradermal administration.